Optimizing direct RT-LAMP to detect transmissible SARS-CoV ...Aug 30, 2020 · Ramuta1, Cecilia G....

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from

primary patient samples

Dawn M. Dudley1, Christina M. Newman1, Andrea M. Weiler2, Mitchell D.

Ramuta1, Cecilia G. Shortreed1, Anna S. Heffron1, Molly A. Accola3, William M.

Rehrauer3, Thomas C. Friedrich4, and *David H. O’Connor1

1Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison,

Madison, WI, USA, 2Wisconsin National Primate Research Center, University of Wisconsin-

Madison, Madison, WI, USA, 3University of Wisconsin Hospitals and Clinics, Madison, WI, USA

4Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI, USA.

*Corresponding author email [email protected]

ORCIDs: DMD (0000-0003-0934-2042), CMN (0000-0002-8749-4378), AMW (0000-0002-7130-8235),

MDR (0000-0003-2134-610X), CGS (0000-0003-0654-5952), ASH (0000-0001-7555-4137), WMR

(0000-0001-8809-1215), TCF (0000-0001-9831-6895), DHO (0000-0003-2139-470X)

1

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https://doi.org/10.1101/2020.08.30.20184796

http://creativecommons.org/licenses/by-nc/4.0/

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from

primary patient samples

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of

nucleic acid extraction supplies and other key reagents have hindered the response to

COVID-19 in the US. Several groups have described loop-mediated isothermal

amplification (LAMP) assays for SARS-CoV-2, including testing directly from

nasopharyngeal swabs and eliminating the need for reagents in short supply. Here we

describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples

and show consistent detection in clinically confirmed samples, albeit with approximately

100-fold lower sensitivity than qRT-PCR. We demonstrate that adding lysis buffer directly

into the RT-LAMP reaction improves the sensitivity of some samples by approximately

10-fold. Overall, the limit of detection (LOD) of RT-LAMP using direct nasopharyngeal

swab or saliva samples without RNA extraction is 1x105-1x106 copies/ml. This LOD is

sufficient to detect samples from which infectious virus can be cultured. Therefore,

samples that test positive in this assay contain levels of virus that are most likely to

perpetuate transmission. Furthermore, purified RNA achieves a similar LOD to qRT-PCR.

These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in

SARS-CoV-2 screening programs, especially while the availability of qRT-PCR testing

and RNA extraction reagents is constrained.

Keywords: RT-LAMP, SARS-CoV-2, NP swab, diagnostic testing, COVID-19, qRT-PCR,

direct LAMP, saliva

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Introduction

There are more than 5.8 million reported severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2) infections in the United States as of August 27, 2020

(https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/cases-in-us.html). The actual

number of infections is likely far greater since diagnostic testing remains limited. We now

understand that asymptomatic individuals contain similar levels of SARS-CoV-2 in the upper

respiratory tract as symptomatic individuals [1–6]. Furthermore, 17 out of 24 (71%)

presymptomatic patients had positive viral cultures 1 to 6 days before the onset of symptoms [1].

Symptom-based screening is not sufficient for controlling SARS-CoV-2 transmission and

emphasizes the need for expanded nucleic acid screening of asymptomatic/presymptomatic

individuals.

Identifying individuals shedding the most SARS-CoV-2 virus is critical for interrupting

transmission. The threshold of virus in a patient sample required to isolate and grow the virus in

tissue culture is one indicator of the viral load necessary to transmit the virus. Recent virological

assessments of COVID-19 patients suggest that virus isolation from patient samples is dependent

on viral load and sample type [1, 7–9]. Wölfel et al. [7] demonstrated that successful SARS-

CoV-2 isolation was limited to only NP swabs and sputum that had viral loads greater than 1x106

copies of viral RNA (vRNA)/ml [7]. This same virus isolation threshold of 1x106 vRNA

copies/ml was also found by Van Kampen et al. [10] when analyzing 129 severe COVID-19

patient samples and Quicke et al. [11] in a longitudinal surveillance study of 454 skilled nursing

facility staff members. During a SARS-CoV-2 outbreak in a Washington nursing home, virus

could be isolated only from NP swabs with RT-PCR cycle threshold (Ct) values of less than 30,

with few exceptions, collected from patients presenting as asymptomatic, presymptomatic, with

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typical and atypical symptoms [1]. A similar virus isolation threshold (Ct value 33-34;

approximately 1x105 RNA copies/ml) was observed in SARS-CoV-2 patients by La Scola et al.

(2020) when using a qRT-PCR assay targeting E gene [12]. In a recent non-human primate

study, virus was successfully isolated from the upper respiratory tract of rhesus macaques

inoculated with 2.6x106 TCID50 of SARS-CoV-2 via a combination of intratracheal, intranasal,

ocular, and oral routes [9]. Positive viral cultures were limited to NP and oral swabs with greater

than 1x106 and 1x105 copies/ml respectively [9]. These data suggest that diagnostic tests with a

sensitivity around 1x106 copies/ml are sufficient for capturing culture-positive cases with the

greatest transmission risk.

Conventional SARS-CoV-2 testing relies on RT-PCR amplification of virus-specific

nucleic acids extracted from nasopharyngeal (NP) swabs. However, shortages of nucleic acid

extraction and RT-PCR reagents as well as RT-PCR instrumentation remain a problem [13].

Alternative nucleic acid extraction methods and "direct" testing that does not require nucleic acid

extraction are important to expand testing while reducing time and cost. Indeed, the SalivaDirect

method recently approved under an FDA EUA, utilizes saliva without RNA extraction into a RT-

PCR assay, eliminating at least part of the process experiencing shortages [14].

Loop-mediated isothermal amplification (LAMP) has been used as a tool for point-of-

need diagnostic testing for several pathogens, including SARS-CoV-2 [15–24]. LAMP assays

are an alternative method for rapidly detecting the presence of specific nucleic acids in samples,

with colorimetric or fluorescent visualization of results. LAMP assays are inexpensive, high-

throughput, do not necessarily require nucleic acid purification, and give rapid results. These

previously published manuscripts demonstrate proof-of-principle for SARS-CoV-2 testing by

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RT-LAMP using either contrived samples with free nucleic acid or primary samples with RNA

isolation first.

In this study, we focused on characterizing and optimizing direct RT-LAMP without

RNA isolation and with primary NP swab samples with known SARS-CoV-2 status. We

demonstrate the limit of detection (LOD) of direct swab RT-LAMP in primary swab samples as

well as modifications of the technique that help improve sensitivity, but don’t rely on the same

materials required for traditional qRT-PCR methods. We characterized the use of Lucigen

QuickExtract (QE) lysis buffer, guanidine hydrochloride addition, an alternative RNA isolation

method, and several primer sets and combinations targeting different gene regions. Lastly, we

used our optimized approach to test the transition to direct RT-LAMP with saliva in a point-of-

need testing approach. Each of these modifications are useful additions to the SARS-CoV-2

testing repertoire and have unique benefits for testing in multiple laboratory settings.

Furthermore, we suggest that the limit of sensitivity achieved with any of these methods is

sufficient to detect levels of virus that can be cultured out of samples and therefore represents

levels where transmission is most likely and self-quarantine most important.

Materials and Methods

Sample collection

Residual NP swab and saliva samples were provided by University of Wisconsin-Madison

Hospitals and Clinics and the Wisconsin State Laboratory of Hygiene under biosafety protocol

B00000117 (IRB 2016-0605) and their use was not considered human subjects research by the

University of Wisconsin-Madison School of Medicine and Public Health's Institutional Review

Board. Samples were collected into a variety of transport media including universal transport

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media (UTM), viral transport media (VTM), and phosphate buffered saline (PBS), stored at 4°C

for up to 7 days, and transported to the laboratory at room temperature. Upon arrival at the

laboratory, samples were stored at either 4°C (for immediate same-day use) or -80°C until use in

RT-PCR or RT-LAMP assays. Residual saliva samples contained no media/buffer and were

stored for 2-4 weeks at 4°C before arriving in the lab and were tested within 2 days of arrival.

qRT-PCR

Viral load analysis was performed after samples arrived in our laboratory. RNA was isolated

using the Viral Total Nucleic Acid kit for the Maxwell RSC instrument (Promega, Madison, WI)

following the manufacturer’s instructions. Saliva samples were diluted 1:1 in water prior to

extraction. Viral load quantification was performed using a qRT-PCR assay developed by the

CDC to detect SARS-CoV-2 (specifically the N1 assay) and commercially available from IDT

(Coralville, IA). The assay was run on a LightCycler 96 or LC480 instrument (Roche,

Indianapolis, IN) using the Taqman Fast Virus 1-step Master Mix enzyme (Thermo Fisher,

Waltham, MA). The LOD of this assay is estimated to be 200 genome equivalents/ml saliva or

swab fluid. To determine the viral load, samples were interpolated onto a standard curve

consisting of serial 10-fold dilutions of in vitro transcribed SARS-CoV-2 N gene RNA kindly

provided by Nathan Grubaugh (Yale University).

RT-LAMP

The experiments we describe here were modified from the SARS-CoV-2 RT-LAMP assay

developed by Zhang et al. [16]. For most assays we used fluorescent-based detection with

Warmstart LAMP reagents and the included fluorescent dye (New England Biolabs, NEB). We

tested primer sets developed in previous studies targeting several SARS-CoV-2 genes as shown

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in Supplemental Table 1 [16, 17, 25–29]. Of note, the Color-Orf1a primers and Lamb-Orf1a

primers are identical, but were used at different concentrations per the protocols developed by

each lab. The final 1X primer concentrations are listed in the Table. For each reaction, a 10X

stock of all 6 primers were combined with Warmstart mastermix and water in 25ul reactions

following the manufacturer’s recommendations. Unless otherwise stated, 1ul RNA transcript of

the SARS-CoV-2 N-gene obtained by Dr. Nathan Grubaugh, 1ul of synthetic SARS-CoV-2 RNA

transcript (Twist Biosciences; RNA control 2), 1ul of gamma-irradiated SARS-CoV-2 (BEI; NR-

52287; isolate USA-WA1/2020), or 1ul primary NP swab sample were tested in each RT-LAMP

reaction. Unless otherwise stated, all serial dilutions were performed in water. For reactions

testing guanidine hydrochloride addition to the RT-LAMP mastermix, a final concentration of

40mM stock was used in the mastermix. Except where otherwise specified, samples were run on

a Roche Lightcycler 96 instrument (Roche Diagnostics) using an 80-cycle program with the

SYBR Green channel at 65°C (495-497 nm absorption; 517-520 nm emission) and data

collection every 30 seconds. For experiments determining the appropriate volume of direct swab

sample addition for highest RT-LAMP efficiency, a 60-cycle program with data collection every

20 seconds was used. For colorimetric RT-LAMP with saliva, saliva was incubated at 65°C for

30 minutes followed by 98°C for 3 minutes in a heat block. Saliva was then diluted 1:1 in PBS

and 3ul was added into a 20ul RT-LAMP mastermix containing the Color-N primer set and

colorimetric mastermix (NEB) and incubated at 65°C for 30 minutes.

Sample lysis

A subset of samples were treated with LucigenQE RNA Extraction Solution (Lucigen,

Middleton, WI) in a 1:1 ratio as described in Ladha et al. [30]. Briefly, NP swab eluate was

combined with an equivalent volume of LucigenQE and briefly vortexed. Samples were then

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incubated for 5 minutes at 95°C, cooled on ice, and maintained until addition to the RT-LAMP

reaction.

Statistical analysis

To assess improvement in quantification cycle (Cq) values using sample lysis with LucigenQE

or RNA isolation, mean Cq values were calculated for each sample. Mean Cq values were not

normally distributed for either dataset so we used a nonparametric equivalent to a paired t-test,

the Wilcoxon signed rank test with continuity correction, for each set of paired samples.

To examine whether sample vRNA load and/or treatment were significantly associated

with a positive RT-LAMP result, we used logistic regression with RT-LAMP result as the

dependent variable. For our analysis, an equivocal result in which one replicate was positive

while the other was negative, was conservatively treated as negative. We coded RT-LAMP

results for each sample tested by each method as a dichotomous outcome with positive samples

coded as “1” and negative or equivocal samples coded as “0”. For explanatory variables, we

chose qRT-PCR vRNA load, with samples greater than 106 copies/ml coded as “1” and samples

less than 106 copies/ml coded as “0”, and group, designated as either 5ul RNA, 1ul lysed, or 1ul

direct addition.

All statistical analyses were performed in RStudio (v. 1.2.1335) using R (v. 3.6.0) [31].

Results

Limit of detection with RNA transcript and Gene-N-A primers

To determine a limit of detection for the RT-LAMP assay, serial 10-fold dilutions of RNA

transcript containing the N-gene were tested in RT-LAMP reactions with Gene-N-A primers in

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duplicate in 3 independent assays. RNA transcripts were diluted in RNase-free water. Consistent

detection of RNA was achieved when 1x106 copies or greater of RNA/ml was added into the

reactions (1x103 copies/ul per reaction) (Figure 1A). To obtain a more precise LOD, transcript

was diluted 1:2 starting at 5x106 copies/ml down to 7.8x104 copies/ml and each concentration

was run in 10 replicates. Nine of ten replicates at 6.25x105 copies/ml were positive, while 6/10

were positive at 3.12x105 copies/ml and 5/10 were positive at 1.56x105 copies/ml (Figure 1A).

Thus, we can consistently detect 625 copies of input into the reaction, but can detect down to 156

copies of input in half of the reactions. Zero reactions were detected as positive at 1x105

copies/ml (100 copies/reaction) or below.

Limit of detection with primary nasopharyngeal swab samples

Leftover NP swab samples from 106 patients with known clinical diagnosis of SARS-CoV-2

were tested directly by RT-LAMP in duplicate. Additionally, RNA was isolated from these

samples and tested by qRT-PCR with a transcript standard for quantitation. A total of 63/106

(59%) samples tested positive by RT-LAMP and 106/106 by qRT-PCR (Supplemental Table 2).

Another 13 samples were equivocal by RT-LAMP, with one of two replicates positive. As shown

in Figure 1B, the LOD of primary samples was similar to that seen with RNA transcript. 63/77

(82%) samples with viral RNA copy numbers greater than 1x106 copies/ml were detected by RT-

LAMP, whereas 0/28 samples with concentrations <1x106 were detected positive and 3/28 were

equivocal. All 31 samples that tested negative for SARS-CoV-2 by clinical laboratories also

tested negative by RT-LAMP (data not shown).

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RT-LAMP is inhibited by adding larger volumes of primary sample

To determine whether adding larger volumes of primary NP swab samples could improve the

sensitivity of the assay, 1, 3, and 5ul of swab samples from 5 primary SARS-CoV-2-positive

samples were tested side-by-side. All replicates were detected as positive when 1ul was added

directly into the RT-LAMP reaction (Figure 1C). However, one of the two replicates from two

samples tested negative when 3ul of sample was added and both replicates from one sample

tested negative when 5ul of sample was added into the RT-LAMP reaction. Furthermore, Cq

thresholds were higher with addition of higher volumes of sample. Therefore, we chose to use

1ul of straight swab sample in subsequent experiments.

Lysis buffer improves the sensitivity of RT-LAMP

To determine whether treatment with lysis buffer improves the sensitivity of RT-LAMP to detect

SARS-CoV-2 in clinical samples without the use of traditional nucleic acid isolation methods,

we treated 72 clinical samples with a range of SARS-CoV-2 vRNA loads in a 1:1 ratio with

LucigenQE as described by Ladha et al. [30]. We then compared the fluorescent RT-LAMP Cq

values between 1ul of lysed sample and 1ul of the same samples added directly. Addition of 1ul

of NP swab eluate directly into the RT-LAMP reaction resulted in positive detection in both

replicates of 46/72 (64%) known SARS-CoV-2-positive samples and in 1 of 2 replicates in 6

additional samples (Figure 2A, Supplemental Table 2). Treatment of the same 72 samples with

LucigenQE resulted in detection of both replicates for 56/72 (78%) samples, an additional 10

samples that were undetectable before. An additional 4 samples that were negative when tested

directly were equivocal when treated with LucigenQE. Negative samples with either method

occurred in samples with viral loads ranging from 1.0x104-6.19x108 vRNA copies/ml. However,

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while 0/19 samples with viral loads below 1x106 were positive with 1ul straight, 4 of 19 samples

were detectable after lysis in both replicates and 4 additional samples were detected in 1 of 2

replicates. Mean Cq for LucigenQE-treated samples were significantly lower (Cq = 38.62) than

those for directly added samples (Cq = 49.08) (Wilcoxon signed rank test, V = 1830, p = 1.67E-

11), suggesting that lysis treatment improves the efficiency of amplification in the LAMP reaction

(Figure 2A). We also examined whether sample vRNA load and/or treatment were significantly

associated with RT-LAMP detection results. We found that direct addition of 1ul of untreated

NP swab was associated with a decreased odds of detecting a positive RT-LAMP result (OR =

0.20, 95%CI = 0.04-0.65, p = 0.015) while the most important factor associated with a positive

result was a sample vRNA load of greater than 106 copies/ml (OR = 88.5, 95%CI = 25.74-

434.87, p = 1.88E-10).

RNA isolation improves the limit of detection of RT-LAMP to levels similar to qRT-PCR

One of the primary reasons the direct RT-LAMP assay is less sensitive than the diagnostic qRT-

PCR assay is because the qRT-PCR assay uses 5ul of concentrated and purified RNA as input.

To determine whether the RT-LAMP assay would perform to a similar level of detection if the

same input was used, 5ul of purified RNA was used in RT-LAMP assays from a subset (n=44) of

COVID-19-positive NP swab samples. The viral loads ranged from 1.01x104 to 1.14x1010. 19 of

44 had concentrations of virus below the RT-LAMP LOD of 1x106 vRNA copies/ml. Of the 44

samples tested with 1ul of direct swab, 18 tested positive (43%) and 7 were equivocal between

replicates (Figure 2B and Supplemental Table 2). When 5ul of purified RNA was used in the

reactions instead, 42 of 44 samples (95%) tested positive (Figure 2B, Supplemental Table 2).

Furthermore, samples with 100-fold lower vRNA copies/ml were detected after RNA isolation

by RT-LAMP. Adding purified RNA brings the possible LOD of detection down to 50 copies of

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input of a 10 copy/ul sample (1x104 copies/ml), which was detected in 4 of 6 primary samples

tested within this viral load range. Overall, the addition of 1uL NP swab direct was associated

with reduced odds of a positive RT-LAMP result (OR = 0.0054, 95%CI = 0.00023-0.041, p =

2.56E-05). Similar to the results for lysis buffer treatment, samples with qRT-PCR vRNA loads

greater than 1x106 copies/ml had significantly increased odds of a positive RT-LAMP result (OR

= 49.35, 95%CI = 8.22-966.29, p = 0.00044). Lastly, the Cq values were lower for all detected

samples when 5ul of RNA was added (mean Cq = 31.42) instead of 1ul straight sample (mean

Cq = 58.72), indicating faster and more robust detection with concentrated and purified RNA

(Wilcoxon signed rank test, V = 903, p = 1.71E-08) (Figure 2B).

Alternative primers improve efficiency

Multiple SARS-CoV-2 RT-LAMP primer sets have been published or included in manuscripts

on pre-print servers since we began our experiments. We compared the efficiency of several

alternative primer sets either alone or in combination to the Gene-N-A primers used in our initial

studies (Supplemental Table 1). Two primary NP swab samples with high concentrations of

SARS-CoV-2 (NP1:1.09x1012, NP2:4.28x1010) as well as gamma-irradiated SARS-CoV-2 (BEI)

were used to screen different primer combinations using the same fluorescent RT-LAMP

conditions. First, a set of primers previously established [17, 27] and used to obtain a FDA EUA

by Color Genomics was tested with each primer alone and in different combinations (Figure 3A).

Several primers and primer combinations resulted in a lower Cq value across the board than the

Gene-N-A gene primers suggesting improved reaction efficiency. The primer combinations

including Color-N/Color-E, and Color-N/Color-ORF1a yielded the lowest Cq values in all

samples.

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Next, we compared the Gene-N-A, Color-N, Color-E, and Color-N/E combination to

second generation primers described in Zhang et al. [25] (Gene-N2, Gene-E1 and As1e). We

found that As1e, originally published by Rabe et al. [26] yielded the lowest Cq value followed

closely by a combination of As1e with the two primers targeting the Gene-N2 and Gene-E1

genes designed by Zhang et al. (Figure 3B). The Color-N primer yielded similar Cq values to the

triple combination primer set from Zhang et al. We also tested the Color-N, Color-E1, and Gene-

N-A gene primer sets against additional published primers that target ORF1a (Lamb, Yu, El-

Tholoth, and Zhang primers) either alone or in combination and compared them to the Gene-N-A

gene primer set (Supplemental Table 1). The Color-N primer produced the lowest Cq value in

this set (Supplemental Figure 2).

We then tested As1e, Color-N, As1e/Color-N/Gene-E1 primer set with and without

guanidine hydrochloride, as recommended by Zhang et al., with Twist RNA SARS-CoV-2

template. Under these conditions, guanidine hydrochloride improved detection with most primer

sets with the exception of the Gene-N-A primer set (Figure 3C). Using the Twist RNA, the

primer set that detected samples at the lowest dilutions was the Color-N primer or combination

of As1e/Color-N/Gene-E1 with guanidine hydrochloride, though only in one of two replicates.

The As1e primer with and without GuHCl as well as the combination As1e/Color-N/Gene-E1

with GuHCl often yielded the lowest Cq value.

Lastly, to determine which primer set or combination worked best with primary NP swab

samples, fourteen samples with viral loads ranging from 7.33 x107-1.52 x1011 vRNA copies/ml

were tested using 1ul of straight swab sample with the most promising primer combinations with

and without GuHCl. Both the Color-N primer set alone and the As1e/Color-N/Gene-E1

combination yielded the lowest Cq value across the different levels of virus (Figure 4A).

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Guanidine hydrochloride did not improve detection in primary samples as seen with the Twist

RNA. Twelve additional primary NP swab samples with high Ct values ranging from 25 to 35

were tested with the Color-N and As1e/Color-N/Gene-E1 combination. Detection of these

samples was very similar between the two primer conditions (Figure 4B).

Transition to saliva-based direct RT-LAMP

To best accommodate point-of-need testing, saliva-based tests using colorimetric read-out

minimizes expensive equipment and eases sample collection. We modified our direct RT-LAMP

approach to accommodate detection of SARS-CoV-2 from saliva while also incorporating heat

inactivation of the virus for safety in the field [32]. Leftover paired NP swab and saliva samples

were tested by their respective RT-LAMP protocols as well as by qRT-PCR (Figure 5).

Detection of SARS-CoV-2 by RT-LAMP was dependent on the viral load of the sample rather

than the starting sample material. Overall, samples with viral loads above 5x105 were detected as

positive by RT-LAMP from either NP swab or saliva. Viral loads from NP swab samples were

often higher than those in saliva in this sample set. This is likely due to the saliva samples being

stored for 2-4 weeks at 4°C before use in this assay while NP swabs were stored for 1-2 weeks at

4°C and then were frozen before use. This data should not be used to assess differences in viral

load observed between saliva and NP swabs because of the differential storage of these leftover

samples. This data does provide proof-of-concept that saliva can be added directly to

colorimetric RT-LAMP reactions and yields a LOD similar to NP swab samples.

Discussion

Frequent, widespread testing is considered the best mitigation strategy to control SARS-CoV-2

before a vaccine or effective therapeutic is available. Traditional qRT-PCR is relatively sensitive,

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but it is time consuming and reliant on very specific reagents that are in short supply in the

ongoing pandemic. RT-LAMP has become a promising alternative to qRT-PCR, but the true

sensitivity of this assay has been poorly characterized from primary NP swab samples when

added directly into the reaction. Many published studies establish a LOD based on free RNA

transcript or isolated RNA from cultured virus of around 100 copies/rxn. These LODs apply to

RT-LAMP only when purified RNA is used as input. They do not apply to direct RT-LAMP

methods containing whole virions in primary samples also containing host enzymes and other

host components. In this study we established that 625 copies/reaction was necessary in order to

detect RNA transcripts consistently in 9/10 reactions in our assay. In primary samples we rarely

detected virus in samples with a vRNA load of less than 5x105-1x106 vRNA copies/ml,

establishing this threshold as a conservative LOD. While not as sensitive as methods with RNA

extraction first, this LOD range is sufficient for SARS-CoV-2 surveillance to detect virus in

individuals with the minimal amount of virus necessary to isolate the virus and therefore most

likely to transmit the virus.

The LOD of 1x106 copies/ml is likely due to inefficiencies associated with virus lysis at

65°C during the LAMP reaction and possible degradation of liberated RNA by enzymes,

including RNases, present in primary samples. Indeed, adding more sample, including more host

enzymes and media with potential inhibitors, reduced detection. On the other hand, lysis with

LucigenQE was compatible with RT-LAMP and improved our sensitivity to detect SARS-CoV-2

in primary samples with vRNA loads less than 106 copies/ml. When compared with direct

addition, we were able to increase our detection of true positives for both replicates from 61% to

78% after lysis treatment. Guanidine hydrochloride has also been shown to improve sensitivity in

other studies and while our results showed better detection with synthetic Twist RNA, bringing

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the LOD down to 62.5 copies/µl, the same improvement was not observed in primary samples

with intact virus. The largest increase in sensitivity occurred with RNA isolation prior to RT-

LAMP using an alternative RNA isolation method to those approved for the CDC qRT-PCR

assay. With RNA isolation we detected 95% of the samples detected by qRT-PCR, including

those with the lowest viral loads.

There are now many primers available targeting different regions of SARS-CoV-2. In

this work we tested several sets to iteratively choose which primer set worked best with primary

NP swab samples. We found that several primer sets performed more efficiently than the Gene-

N-A primers we used in most of our experiments. The two best performing primer sets that were

nearly indistinguishable in performance with both high and low viral load primary samples were

a combination of As1e/Color-N/Gene E1 and the Color-N primer set alone.

For most of this study we chose to use fluorescent RT-LAMP for detection rather than

colorimetric detection. Using fluorescence enabled detection of the differences in the Cq values

providing a quantitative evaluation of how each condition changed efficiency of the assay.

However, when considering how to deploy RT-LAMP in the field, our group has developed a

mobile RT-LAMP workflow that uses saliva and colorimetric readouts for low cost and

portability. Additional work needs to be done to determine whether the benefits of fluorescent

detection can be inexpensively migrated to decentralized point-of-need testing.

Many studies are comparing RT-LAMP to RT-PCR results presented as Ct value, rather

than vRNA copies/ml. We chose to focus on comparing methods and determining LODs based

on a standard qRT-PCR assay performed in our laboratory with a quantitative standard, rather

than Ct values generated by the varying sources of our primary samples. The hospital

laboratories have transitioned between different methods as reagents were available and as new

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assays became available, which means that the Ct values we obtained for each of our samples

was generated by different assays targeting different regions of the SARS-CoV-2 genome. By

comparing all our results to the vRNA copies/ml that we generated in our lab using a consistent

primer set and protocol (CDC qRT-PCR assay) that targeted the same gene (and primers) as our

SARS-CoV-2 RT-LAMP assay, we were able to ensure our comparisons were consistent across

all samples.

Overall, we have shown that direct RT-LAMP using fluorescent detection can detect

SARS-CoV-2 in primary NP swab samples with viral loads greater than 1x106 vRNA copies/ml.

We were able to improve this slightly with the quick and low-cost addition of Lucigen lysis

buffer to the reaction. We also saw improvement in efficiency with several alternative primer

sets. While direct RT-LAMP is not as sensitive as qRT-PCR, the gold standard for diagnosis, it

is sufficient to detect the levels of virus that are necessary to culture virus from a sample as

described previously. This means that this assay detects people who are likely to transmit the

virus for a significant reduction in cost, time, and reagents relative to qRT-PCR. Lastly, we

confirmed that saliva was also compatible with direct RT-LAMP assays when heated first.

Overall, direct RT-LAMP is an important addition to the repertoire of currently available tests to

identify samples containing SARS-CoV-2 nucleic acids.

Acknowledgements

We thank Nathan Tanner for discussions about primers and optimizations of the RT-LAMP

assay. The following reagent was deposited by the Centers for Disease Control and Prevention

and obtained through BEI resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-

WA1/2020, Gamma-Irradiated, NR-52287.

17



https://doi.org/10.1101/2020.08.30.20184796


Declaration of interests

The authors disclose no potential competing interests. This work was supported in part by the

Office of Research Infrastructure Programs/OD under grant P51OD011106 awarded to the

Wisconsin National Primate Research Center at the University of Wisconsin-Madison, the Rapid

Acceleration of Diagnostics (RADX) program through the National Institutes of Health (contract

number pending), and by the Wisconsin Alumni Research Foundation.

Data availability

All data supporting the results are presented either in the main paper or as part of the

supplemental online material. Additional details about protocols and results are posted at

https://openresearch.labkey.com/Coven/wiki-page.view?name=lamp-testing.

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C. 1ul 3ul 5ul

1 2 3 4 530

40

50

ND

RT-

LAM

P C

q

A.

B.

103 104 105 106 107 108 109 1010 10110

20

40

60

ND

vRNA copies/ml

RT-

LAM

P C

q

103 104 105 106 107 108 109 10100

20

40

60

ND

vRNA copies/ml

RT-

LAM

P C

q

Figure 1: Detection of SARS-CoV-2 by RT-LAMP from transcript or primary NP swab samples. A. The quanti-fication cycle (Cq) relative to each transcript copy number is plotted. Samples that were not detectable were plotted on the line labeled ND for all graphs at Cq of 60 or 80, the total number of cycles run in our assays. The vertical line is set at the lowest dilution where positive samples were detected using the transcript input for all graphs (156,300 vRNA copies/ml). Each replicate is plotted on all graphs. B. Detection of 106 SARS-CoV-2 positive primary NP swab samples relative to their in-house viral load value. C. RT-LAMP Cq of five SARS-CoV-2 positive primary NP swab samples with different swab input volumes.

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A.LucigenQE 1uL straight

103 104 105 106 107 108 109 1010 10110

20

40

60

ND

vRNA copies/ml

RT-

LAM

P C

qB.

5ul vRNA 1ul straight

103 104 105 106 107 108 109 1010 10110

20

40

60

ND

vRNA Copies/ml

RT-

LAM

P C

q

Figure 2: Comparison of the detection of SARS-CoV-2 positive primary NP swab samples after using direct sample addition to either LucigenQE treatment or isolated vRNA. The vertical line is set to the lowest con-centration detected using RNA transcript as input into the RT-LAMP reaction for reference in all graphs. Samples that were not detectable were plotted on the line labeled ND at a Cq of 80. A. Comparison of Cq values between samples treated with or without LucigenQE and run by RT-LAMP with 1ul of sample. B. Comparison of Cq values between RT-LAMP assays run with 1ul straight swab or 5ul of isolated and puri-fied vRNA.

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https://doi.org/10.1101/2020.08.30.20184796


Gene-N-A

Color-N

Color-E

Color-ORF1a

Color-RNaseP

Color-N/E

Color-N/ORF1a

Color-E/ORF1a

Color-N/E/ORF1a

NP1 NP2 IR SARS-CoV-20

10

20

30

40

5060

ND

RT-

LAM

P C

q

As1e

Gene-E1Gene-N2

Gene-N-A

Gene-N2/E1Gene-N2/E1/As1e

Color-NColor-E

Color-N/E

NP1 NP2 IR SARS-CoV-20

10

20

30

40

5060

ND

RT-

LAM

P C

q

As1e

As1e + GuHCl

Color-N

Color-N + GuHCl

As1e/Color-N/Gene-E1As1e/Color-N/Gene-E1 + GuHCl

Gene-N-A

Gene-N-A + GuHCl

A.

B.

C.

7 15 31.25 62.5 125 250 500 1000 1000015

20

25

30

35

40

45506070ND

SARS-CoV-2 Twist RNA (copies/μL)

RT-

LAM

P C

q

Figure 3: Comparison of RT-LAMP Cq value on primary NP swab samples, irradiated SARS-CoV-2 or SARS-CoV-2 TWIST RNA amplified with different primer sets. Reactions were run in duplicate and both replicates are shown on the graphs. Samples that were not detectable are plotted on the ND line at Cq 80. A. Comparison of Cq values using Color Genomics primers to the Gene-N-A primers on two primary NP swab samples and irradiated SARS-CoV-2. B. Comparison of Zhang et al.{ 32635743} primers to a subset of Color Genomics primers and Gene-N-A primers on two primary samples and irradiated SARS-CoV-2. C. Comparison of the Cq values obtained when using the best primers and combinations of primers across different dilutions of SARS-CoV-2 TWIST RNA with and without GuHCl.

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Color-NAs1e/Color-N/Gene-E1

As1e/Color-N/Gene-E1

Color-N

Gene-N-A

As1e/Color-N/Gene-E1 + GuHCL

Color-N-GuHCL

Gene-N-A+ GuHCL

A.

B.

106 107 108 109 1010 1011 101210

20

30

40

50

60ND

vRNA copies/ml

RT-

LAM

P C

q

20 25 30 35 4010

20

30

40

50

60ND

Ct, CDC qRT-PCR

RT-

LAM

P C

q

Figure 4: Comparison of the best-performing primers and combinations on additional primary NP swab samples with varying levels of virus with and without GuHCl. A. Comparison of three primer sets or combinations either with or without GuHCl on 14 primary NP swab samples with high viral load copy numbers. B. Comparison of the Color-N primer set to As1e/Color-N/Gene-E1 on primary NP swab samples with lower levels of virus. These samples were not run by our in-house viral load test and therefore Ct value obtained from the hospital is reported.

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Saliva NP swab

4.94E+7 2.58E+6 3.36E+4 2.56E+7

1 2 3 4

8.20E+7 1.20E+7 3.14E+8 2.51E+3

4.72E+7 7.89E+3 9.57E+6 8.33E+6

6.20E+5 5.49E+7 8.88E+6 1.49E+4

4.33E+3 1.74E+6 -C saliva -C water 2.10E+4 3.14E+7 - C water +Control

3.99E+3 negative5.96E+6 4.95E+6

9.48E+5 4.40E+76.32E+6 6.12E+2

1.13E+5 2.10E+7 5.72E+4*7.96E+2

+Control1.31E+5 5.41E+4 2.58E+6

-C water

+Control

3.16E+7 3.30E+8

2.35E+3 4.29E+7 2.28E+8

4.93E+6 5.04E+4 2.43E+8

1 2 3 4

5 6 7 8 5 6 7 8

9 10 9 10

11 12 13 14 11 12 14 15

15 16 17 18 16 18 19 20

19 20 21 22 21 22 25

23 24 25

1.72E+5 9.34E+2

4.07E+2

Figure 5: Colorimetric RT-LAMP from SARS-CoV-2 positive paired NP swab and saliva samples. Positive samples are yellow and negative samples are red. qRT-PCR-based viral loads are presented above each sample pair. Paired numbers are from the same patient. Some saliva samples did not have paired NP swab samples. Irradiated SARS-CoV-2 was used as a positive control.

27



https://doi.org/10.1101/2020.08.30.20184796


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